Recent interest in biofuels and alternative sources of chemicals has made microbial production of bulk chemicals an important focus of development. However, many chemicals of industrial interest are toxic to the microorganisms producing them. Microbial production levels are thus constrained by the toxicity limits of the organism to the final product. Host engineering to improve strain tolerance towards target compounds is thus important to increase production levels.
Different compounds may require different host engineering approaches, as toxicity relates to the mode of inhibition, chemical properties, and hydrophobicity of a compound. Hydrophobic solvent-like compounds are hypothesized to impact membrane permeability and fluidity, diminish energy transduction and interfere with membrane protein function, affecting a range of essential cellular processes (Wagner et al, 2007). The cell defense mechanisms that respond to these compounds include induction of chaperones, modification of membrane composition and cellular morphology, and induction of active membrane efflux pump transporter portents that export the compounds out of the cells and the membrane. Of these, efflux pumps have recently emerged as an important target in engineering of host cells (Doshi et al. 2013; Dunlop et al. 2011; Dunlop et al, 2010; Fisher et al. 2013; Foo and Leong 2013).
Efflux pump proteins, also referred to herein simply as “pump” proteins, provide the general mechanism for the export of toxic compounds from cells (Nikaido 2009: Takatsuka et al. 2010). One of the best-characterized families of pumps, and also the key pumps in gram negative bacteria for tolerance towards solvent-like compounds, belong to the hydrophobe/amphiphile efflux (HAE1) family of resistance-nodulation-di vision (RND) pumps (Nikaido and Takatsuka 2009; Ramos et al. 2002; Tseng et al. 1999). RND efflux pumps are composed of 3 subunits: (i) an inner membrane unit proton antiporter that binds the substrate and transports if through (ii) the outer membrane channel subunit, and (iii) periplasmic subunit that connects and stabilizes the inner and outer membrane units (Nikaido and Takatsuka 2009).
The E. coli AcrAB-TolC efflux pump is a member of the HAE1 family. AcrAB-TolC is composed of AcrB (inner membrane protein), TolC (outer membrane protein) and AcrA (periplasmic protein) and has been extensively studied (Murakami et al. 2002; Tikhonova et al. 2011). There are many reports in the scientific literature characterizing the mechanism of action of AcrB, including its rotational conformation changes (Seeger et al. 2006; Seeger et al. 2008; Sennhauser et al. 2007; Takatsuka and Nikaido 2009; Takatsuka and Nikaido 2010) as well as potential binding pockets and the amino acids involved in substrate recognition (Eicher et al. 2012; Husain and Nikaido 2010; Vargiu and Nikaido 2012). The reported substrate entry points have been located in the periplasm (Takatsuka and Nikaido 2007), the membrane and/or in the cytoplasm side of the pump (Eicher et al. 2012; Husain and Nikaido 2010; Murakami et al. 2004; Murakami and Yamaguchi 2003; Sennhauser et al. 2007). Although AcrB functions with AcrA and TolC in E. coli, AcrB also has efflux pump activity in the absence of AcrA and TolC (see, e.g., Kapoor & Wendell, Nano Lett. 2013, 13, 2189-2193, 2013).
AcrB has broad substrate specificity that ranges from detergents to antibiotics and solvents. This pump is reported to play a major role in the secretion of various alkanes such as hexane, heptane, octane, and nonane (Takatsuka et al. 2010) and also in imparting tolerance to various terpene based biofuel compounds (Dunlop et al. 2011).
Production of α-olefins by microorganisms is hampered by toxicity of the compound to the host cells. There is therefore a need to improve tolerance. This invention addresses that need, in part, by providing host cells with genetic modifications to an acrB gene, or homolog, that result in improved tolerance to α-olefins and accordingly, increased α-olefin yields from modified host cells that produce the compounds.